Imidazoline receptors are now generally recognized as a unique set of non-adrenergic high affinity binding sites for a number of agents that to date also bind to α2-adrenergic receptors (Eglen, R. M. et al., 1998, Trends in Pharmacol. Sci. 19:381-390; Regunathan, S. and Reis, D. J., 1996, Ann. Rev. Pharmacol. Toxicol. 36:511-44). Although only one imidazoline receptors has been cloned, evidence including differences in selectivity and binding affinity of ligands, the structure of binding proteins, cellular distribution and activities indicate that they are different from α2-adrenergic receptors. The nonadrenergic imidazoline receptors are important in mediating the hypotensive actions of clinically important imidazoline drugs such as clonidine, rilmenidine and moxonidine.
For example, unique imidazoline receptors, are present in pancreatic islet and beta-cells (Morgan, N. G., et al., 1995, Ann. N.Y. Acad. Sci. 763:361-373). Activation of these receptors by imidazolines causes release of insulin. Some of this activity may be due to imidazoline-induced closure of K+ channels such as the K+ ATP-sensitive channels which permits intracellular levels of K+ to accumulate, causing cell depolarization and eventual exocytosis of hormone or transmitter into plasma or extracellular fluid. It is noteworthy that channels such as the K+ ATP-sensitive channels exist throughout the body, and are particularly abundant in brain. These pancreatic imidazoline receptors have recently been designated as I3 receptor subtypes (Eglen, R. M. et al., 1998, TIPS 19:381-390). However, the I3 subtype may not be linked to K+ ATP channels. Chan et al. (1997, Brit. J. Pharmacol. 120:926-932), showed that imidazolines and preparations of CDS (clonidine-displacing substance, see below) from bovine brain caused release of insulin and stimulated K+ ATP channels.
One or more endogenous ligands selectively bind to the imidazoline receptors although attempts to identify this endogenous ligand(s) have failed. A possible ligand, referred to as “clonidine displacing substance” (CDS), has been discovered as an entity isolated from mammalian brain and the periphery that is capable of displacing radio-labeled clonidine and its radio-labeled congeners from membranes (Atlas, D. et al., 1987, J. Cardiovascular Pharmacology 10 (Suppl. 12): S122-S127; Atlas, D. 1991, Biochemical Pharmacology 41:1541-1549; Atlas, D., 1995, Annals of the New York Academy of Sciences 763:314-324). Antibodies have been prepared against the drug clonidine, which presumably interact with CDS. Such antibodies are found to be immunoreactive in tissues throughout the body and also show a heterogeneous regional distribution within the brain.
A recent study proposed that agmatine, a known compound isolated from bovine brain, is CDS (Li, G. et al., 1994, Science 263:966-968; Regunathan, S. and Reis, D. J., 1996, Ann. Rev. Pharmacol. Toxicol. 36: 511-544; but see Eglen, R. M. et al., 1998, TIPS 19:381-390). Agmatine was further suggested to be an endogenous neurotransmitter because it was found within an extract of CDS activity from whole brain and because it appeared to bind to a class of imidazoline receptors. However, comparisons of the biological activities of agmatine, e.g., effects on blood pressure versus effects of endogenous clonidine-displacing substance at imidazoline and α2-adrenergic receptors produced in virtually all laboratories, indicated that agmatine differed from “classical CDS.” For example, agmatine displaces labeled clonidine from a subset of its nonadrenergic binding sites identified as imidazoline 2A and 2B sites. However, because those I2A and I2B sites are now known to be enzymes, i.e. portions of monoamine oxidase A and B, the search for the identity of CDS that acts at membrane-bound imidazoline receptors has continued (Eglen, R. M. et al., 1998, TIPS 19:381-390).
Several laboratories have harvested CDS and most preparations show similar physiochemical properties. There is widespread consensus that CDS is present in small amounts in the brain, cerebrospinal fluid and periphery (including plasma) of mammals. It is soluble in water and methanol, but generally insoluble in organic solvents. Size exclusion chromatography indicated that it is a small molecule (≦1000 Da). CDS is resistant to several proteases, including trypsin and chymotrypsin, and is devoid of amino acids; thus it is not a peptide. CDS appears to have no free amino groups as activity is retained following reaction with fluorescamine and ninhydrin. CDS is stable in both weak acids (pH 2) and weak bases (pH 10.5), is thermostable (at 110° C.) and retains activity following multiple freeze-thaw and lyophilization cycles. Because CDS can be retained on both anion and cation exchange resins and because its migration patterns shifted markedly with changes in ambient pH on gel electrophoresis, it is very likely that CDS is amphoteric, possibly a zwitterion. In addition, CDS shows maximal UV absorbance between 206-220 nm.